Throughout this application various publications are referenced to by arabic numerals within parenthesis. Full bibliographic citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures for the publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
It has long been known that dietary restriction of vitamin A causes widespread abnormalities in tissue and organ physiology, especially in neonates. The vitamin A deficiency syndrome is characterized by generally stunted growth, keratoses of skin and eyes (1) (leading in severe cases to blindness), defective testis development (1) etc., and atrophy of central (i.e., thymus and bursa of Fabrizius) and peripheral lymphoid organs. Consequently, immune functions are severely affected. Even in mild cases of vitamin A deficiency, the immune system appears to be hyporesponsive. In a recent study in southern India (2), the authors noted in children suffering from mild vitamin A deprivation significantly higher mortality rates in common childhood diseases compared with children receiving normal dietary levels of vitamin A. Since severity but not susceptibility to infection was correlated with vitamin A deprivation, it is likely that reduced immune functions are a factor.
In the absence of retinol, lymphoblastoid cells (LCL) die within 24 to 48 hours (3). Retinol and retinaldehyde, but not retinoic acid, support the growth of LCL in serum-free medium. The same is true for activated human thymocytes. These finding may represent direct correlates to the lagging development of lymphoid organs described by Wolbach and Howe (1) and the in-vivo immune system dysfunction referred to earlier (2).
Nearly all vertebrate tissues are bathed in a constant supply of vitamin A, and the ubiquitous distribution of cellular retinol-binding protein (CRBP) with its high affinity to retinol suggests that it is inside most cells as well. Yet the general purpose of retinol, its metabolism and final destination, remain for the most part unknown, the well-studied example of specialized usage such as vision notwithstanding. Since retinal is not known to be incorporated into structural parts of cells and does not bind to one of the yet analyzed transcription factors with high enough affinity, its role is more likely to be found in its function as precursor for derivatives. Use of retinaldehyde in vision is one example, and another that of retinoic acid as a morphogen (4), important for development of limb and brain. When coupled with parallel discoveries of retinoic acid receptors (RAR) (5-7) within the larger steroid receptor superfamily (8), a sound molecular foundation is given. In this hypothesis, RARs bind to specific response elements in the promoter regions of genes. Retinoic acid in turn binds to RAR, causing activation of gene transcription. The universal principle of this genetic control has increasingly been highlighted by observations that many developmentally important genes from drosophila to man are part of the retinoic acid/steroid receptor superfamily. Moreover, for more than two dozen "orphan receptors" (8) engaged in control of the general physiology of cells, the ligands are not known and are suspected to be small lipophilic molecules.
Many mammalian tissues are dependent on a source of retinol for ordered growth and development, and this requirement is also reflected in certain cell types propagated in tissue culture systems. The tissue culture medium needs to be fortified with retinol at a concentration approximating that of serum (i.a., 10.sup.-6 M). It is widely believed that retinol serves as a precursor molecule for production of a number of cellular metabolites that are the true mediators of retinol effects. Examples are retinoic acids (RA, all-trans RA, and 9-cis-RA) that have been implicated in differentiation and gene regulation, respectively.
Lymphocytes also exhibit a dramatic need for retinol as a co-factor in their activation and partly for the maintenance of the proliferative state. These effects on lymphocytes are not mediated by RA. Instead the presumptive mediator has been identified as a new retinoid molecule hitherto unknown in nature that can activate certain physiological processes in lymphocytes. This compound is 14-hydroxy-4,14-retro-retinol (14-hydroxy-retro-.alpha.-retinol) (14HRR), which may work along a pathway parallel to the well established retinoic acid pathway, but leading to distinct physiological responses.
This compound is synthesized by B cells, T cells and a variety of other mammalian and insect cells. 14HRR is as active (in T cells) or 10-30 fold more active on as concentration basis in B cells as all-trans retinol. Evidently, 14HRR functions as a new type of second messenger molecules.
These findings prompted an inquiry into the biosynthetic pathway of the production of 14HRR. Based upon this inquiry, a new retinoid, previously neither observed in nature or obtained by organic synthesis, has been purified and synthesized. Its structural formula is 13,14-dihydroxy-retinol (13,14 DHR). This compound has been shown to be a metabolic intermediate in 14HRR biosynthesis and is capable of being used in lieu of retinol as a cofactor for activation and growth.